Human amniotic fluid stem cells are able to form embryoid body-like aggregates which performs specific functions: morphological evidences

Lucia Centurione,Maria Antonietta Centurione,Ivana Antonucci,Silvia Sancilio,Roberta Di Pietro,Gianmarco Stati,Liborio Stuppia

doi:10.1007/s00418-020-01940-3

Lucia Centurione, Maria Antonietta Centurione + Show 5 more

Open Access

https://doi.org/10.1007/s00418-020-01940-3

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Abstract

Human second trimester Amniotic Fluid Stem Cells (hAFSCs) harbour the potential to differentiate into cells of each of the three germ layers and to form Embryoid Body (EB)-like aggregates, without inducing teratoma formation and with no ethical concerns. However, in spite of the number of reports on hAFSCs-EBs and their characterization, a thorough evaluation in light and electron microscopy of morphological and morphometric features of hAFSCs-EBs development in vitro has not been reported yet. Apart from a superficial layer of epithelial-like flat cells, displaying rare microvilli on the free surface, hAFSCs-EBs enclose inner material, abundant in vesicles and secretory granules, showing early characteristics of connective extracellular matrix dispersed among different types of inner cells. The observation of a number of microvesicles mainly represented by microparticles and, to a lower extent, by exosomes indicates the presence of a complex cellular communication system within this structure. According to morphological analysis, after 7 days of in vitro culture hAFSCs-EB appears as a well-organized corpuscle, sufficiently young to be a carrier of stemness and at the same time, when appropriately stimulated, able to differentiate. In fact, 7-day hAFSCs-EB represents itself an initial cellular transformation towards a specialized structure both in recording and in providing different stimuli from the surrounding environment, organizing structures and cells towards a differentiation fate.

Highlights

One of the most remarkable properties of Amniotic Fluid Stem Cells (AFSCs) is their capacity to organize themselves into structures that are able to mimic some of three-dimensional (3D) properties of embryonic development
Materials and methods Human second trimester Amniotic Fluid Stem Cells (hAFSCs) collection and culture and Embryoid Body (EB) formation with hanging drop method Briefly, to form EBs hAFSCs at the eighth passage were harvested with 0.05% trypsin and cultured in suspension in IMDM containing 15% FBS, 1 mM glutamine, 0.1 mM β-mercaptoethanol, and 1% penicillin–streptomycin. 25-μl drops (1000–10,000 cells) were plated on the lid of Petri dishes (M-Medical, Milan, Italy), which were inverted onto a dish filled with 8 ml 1X phosphate-buffered saline (PBS; M-Medical) to maintain the humidity
Haematoxylin–Eosin stained sections showed a roundish cellular aggregate (Fig. 3a), surrounded by flattened epithelial-like cells on the surface and containing inside an amorphous rarefied material mixed with elongated fibroblast-like cells, round cells, apoptotic cells and cellular debris (Fig. 3b)

Summary

Introduction

One of the most remarkable properties of Amniotic Fluid Stem Cells (AFSCs) is their capacity to organize themselves into structures that are able to mimic some of three-dimensional (3D) properties of embryonic development. These so-called embryoid bodies represent an experimental model that has provided many important clues for unraveling early embryonic development (Brickman and Serup 2017). An EB is generally accepted as a characteristic self-maintaining pluripotent stem cells aggregate where cells typically “clump together” It is considered as a model of early embryonic development and a feature of pluripotency (Ten Berge et al 2008; Attia et al 2014).

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